Project 3 Summary The intestine is a rich lymphoid organ. The success of intestinal transplantation (ITx) for the treatment of intestinal failure is limited by high rates of rejection and risks of graft-vs-host disease (GVHD) and infection. On the funded grant (Project 4), we addressed the hypothesis that these outcomes were largely determined by the exchange of lymphoid tissue and hence the balance of graft-vs-host (GVH) and host-vs-graft (HvG)-reactive T cells. By combining multiparameter FCM, including allele-specific mAbs to distinguish donor and recipient- derived cells, with high-throughput TCR ? chain CDR3 sequencing to track alloreactive T cells in the GVH and HvG directions in the graft and circulation, we obtained data consistent with this hypothesis. High-level multilineage donor hematopoietic chimerism was common in the recipient blood after transplant, often persisted >1 year and usually was not associated with clinical GVHD. Increased blood chimerism in recipients of multivisceral transplants (MvTx) was associated with reduced donor-specific antibody (DSA)-positive rejection and slower replacement of donor graft T cells by the recipient compared to recipients of isolated intestinal transplants (iITx), demonstrating interrelationships between lymphocytes in the graft and the circulating T cell pool. Our data suggest that GvH-reactive tissue resident memory (TRM) cells in the graft expand locally in response to rapidly infiltrating recipient APCs, then enter the recipient?s peripheral circulation and possibly the bone marrow, where they may promote engraftment of donor graft-derived hematopoietic stem cells (HSCs) and progenitors that contribute to multilineage donor blood chimerism. Early rejection is associated with graft mucosal infiltration by recipient T cells with a non-TRM, circulating T cell phenotype, in which HvG T cell clones predominate. These HvG clones persist within the graft, acquire the TRM phenotype after the rejection resolves, and apparently seed the entire gut, potentially posing a constant threat of rejection. Our data suggest that GVH- reactive clones may counteract HvG reactivity both locally within the graft, thereby protecting it from rejection, and systemically. In this renewal, we will build on these findings and those of Projects 1 and 2 to address the central hypothesis that local antigen responses establish and maintain the intestinal lymphocyte resident memory pools, that TRM and effector T cells (Teff) may be interconvertible and that graft TRM/Teff can enter the circulation and recipient bone marrow. We propose to: 1) Determine the functional and spatial regulation of alloreactive T cell responses in the graft, circulation and bone marrow; and 2) Assess the homeostasis, specificity and genesis of B cell responses in the graft, bone marrow and circulation. Project 3 will provide unique insights into the temporal development of human tissue resident lymphocyte populations that complement the spatial analyses carried out in Projects 1 and 2, as well as a deeper understanding of the immunobiology of transplant rejection and DSA formation.